Worldwide, the vast majority of 16.4 million new infections with HIV are acquired through sexual transmission. Compounds that are developed as topical microbicides to prevent HIV transmission offer a promising alternative while awaiting the development of an effective vaccine. Project 1 will be focused on the optimization of combination topical microbicides targeting different points in the HIV life cycle with the goal of developing a maximally effective and safe formulation for blocking the sexual transmission of HIV-1. The combinations will all include formulated naphthalene sulfate (PRO 2000) presently undergoing Phase II-III evaluation as a single compound as well as a second compound with a unique antiviral mechanism. PRO 2000 predominantly inhibits viral entry by interfering with gp120 interaction(s) with the cell surface. Since this compound is not a virucidal and only works at entry, combining it with compounds that are virucidal and/or work on a separate phase of the virus life cycle should have an additive if not synergistic benefit. Additive and/or synergistic anti-HIV effects of PRO2000 with inhibitors of reverse transcriptase including the non-nucleoside, UC781 and the nucleotide analogue, (R)-9-(2-phosphonylmethoxypropyl)adenine (PMPA or tenofovir), and topical virucidal agents bile salts and amphiphilic polymers will be determined in a single-cycle infection assay. Lead combinations will then be evaluated in primary culture and cervical/vaginal as well as rectal tissue explant cultures in the presence of cervical lavage and seminal fluid. Additional in vitro studies will focus on the generation of viral variants relatively resistant to PRO 2000 which will not only provide critical insights into the potential generation of resistance in vivo but also provide information regarding potential cross resistance with other compounds. Lastly, this project will provide virologic support in the clinical evaluation of women exposed to PRO 2000 as proposed in Project 3. These studies will both identify leading effective combination topical microbicides as well as promising virologic assays to assess their effectiveness.